1. Field of the Invention
The present invention relates to novel pyrylium compounds, methods for making the pyrylium compounds, and nucleic acids labeled with the pyrylium compounds. In particular, the present invention relates to a novel pyrylium compound which can be used as a label when a target nucleic acid is to be detected by optical means, to a process for making the pyrylium compound, and to a nucleic acid labeled with the pyrylium compound.
2. Description of the Related Art
Fluorescent dyes are widely used as labels for detecting target nucleic acids in biological samples. When the fluorescent dye is used as a label, a biological sample and the fluorescent dye are mixed and are allowed to stand in an environment in which the nucleic acid interacts with and links together the fluorescent dye on a molecular level. When the sample contains nucleic acids, the spectroscopic characteristics of the fluorescent dye change with such interactions. By detecting such changes, the nucleic acids in the sample can be identified or quantified. Interactions between the nucleic acids and the fluorescent dyes which have been conventionally used can be categorized as an intercalation in which fluorescent dyes are intercalated into base pairs of nucleic acids or as groove binding in which fluorescent dyes are incorporated along the groove of a double helix.
Examples of spectroscopic changes due to the interaction of the fluorescent dye include a shift of a peak wavelength in an absorption spectrum toward a shorter or longer wavelength, a change (increase or decrease) in absorbance, and a change (increase or decrease) in fluorescence intensity. When a target nucleic acid is detected by using a fluorescent dye as a label, changes (increase and decrease) in fluorescence intensity of the label are often used. Examples of known dyes interacting with the nucleic acids include acridine orange, proflavine, ethidium bromide, donomycin, actinomycin, and pyrylium salts. For example, pyrylium salts interacting with nucleic acids exhibit large shifts of absorption spectra toward longer wavelengths, and adequately large fluorescence intensity compared to the fluorescence intensity of the pyrylium salts in the free state. These two properties are advantageous for labeling of nucleic acids, and thus, the pyrylium salts have attracted attention as dyeing agents or labeling agents for nucleic acids. Many pyrylium salts interacting with nucleic acids have been proposed and synthesized, and properties thereof have been investigated.
One characteristic feature of the pyrylium salts is light absorption in the visible light region of the dyes. Since most of the above fluorescent dyes other than the pyrylium salts fluoresce, the excitation wavelength must be near the ultraviolet (UV) region. Japanese Unexamined Patent Application Publication No. 9-40661 by the present inventors discloses pyrylium compounds which have an absorption peak in a visible light region near 550 nm in an aqueous solvent so that the excitation wavelength is selected in the visible light region. When biological samples are living eukaryotic and prokaryotic cells, they will be damaged by UV irradiation of UV light as excitation light. Thus, the pyrylium salts for which the excitation light can be set within a visible light region are preferable since cell damage can be avoided.
The pyrylium salts have attracted attention as nucleic acid stains or fluorescence labeling agents. As described above, however, current interactions between the nucleic acids and the fluorescent dyes are classified into two types, that is, intercalation in which fluorescent dyes are intercalated between base pairs of nucleic acids, and groove binding in which fluorescent dyes are incorporated along the groove of the double helix. Japanese Unexamined Patent Application Publication No. 9-40661 by the present inventors discloses specific detection of target double-stranded nucleic acids using the above pyrylium compounds, and use of the pyrylium compounds as a probe by binding the compounds to a single stranded nucleic acid. For example, the pyrylium compounds represented by the following formulae (III) and (IV) are bonded to a single-stranded nucleic acid so that a carboxyl group bound to a pyrylium ring through a phenylene group forms an amido bond with an amino group which is incorporated into an end or a part of the single-stranded nucleic acid: 
One of the preferred properties of the probe comprising the pyrylium compound bound to a single-stranded nucleic acid is its ability to maintain its original absorbance and luminescence of the pyrylium compound even after binding to the single-stranded nucleic acid. Although the probes disclosed in the above patent are comparable to the unreacted pyrylium compounds with regard to specific detection of target nucleic acids, shifts in wavelength of the absorbance and the fluorescence were sometimes observed, and in the case where a shift was observed, it is necessary to re-calibrate the fluorescence detector.
Accordingly, it is an object of the present invention to provide a novel pyrylium compound which does not result in changes in optical characteristics, such as absorbance and fluorescence, when the pyrylium compound is bonded to a single-stranded nucleic acid, a process for making the pyrylium compound, and a labeled nucleic acid containing the pyrylium compound as a label.
The present inventors have researched changes in optical characteristics when a pyrylium compound having a carboxyl group which is bound to the pyrylium ring through a phenylene group is bonded to a single-stranded nucleic acid. As a result, the present inventors have hypothesized as follows. When an aromatic ring, such as a p-phenylene group or a 2-(p-phenylene)vinyl group, having a carboxyl group is directly bonded to a pyrylium ring having two 4-N,N-dimethylaminophenyl groups, some nucleic acids to be bonded affect the optical characteristics of the pyrylium pigment. Based on these results, the present inventors have attempted to bond of a pyrylium ring having two 4-N,N-dimethylaminophenyl groups to a carboxyl group with a non-conjugated group such as a methylene group therebetween. The present inventors have discovered that bonding of a single-stranded nucleic acid does not substantially affect the conjugated electronic state of the pyrylium ring and that the optical characteristics, such as fluorescence, of the unbonded pyrylium compound are maintained. The present invention has been accomplished according to these results.
According to an aspect of the present invention, there is provided a pyrylium compound represented by the following formula (I): 
wherein X is oxygen or sulfur, Yxe2x88x92 is a monovalent anion, n is an integer of 2 or 3, and M is hydrogen or an alkali metal.
According to another aspect of the present invention, there is provided a nucleic acid stain comprising the pyrylium compound represented by the formula (I).
According to another aspect of the present invention, there is provided a process for making the pyrylium compound represented by the formula (I), wherein X is oxygen, comprising the step of subjecting a cyclic anhydride represented by the following formula (II) to a condensation-cyclization reaction with 4-N,N-dimethylaminoacetophenone: 
wherein n is an integer of 2 or 3.
According to a further aspect of the present invention, there is provided a labeled nucleic acid which is obtainable by reacting the pyrylium compound according to the above aspects with a single-stranded nucleic acid having a functional group capable of reacting with a carboxyl group.
According to another aspect of the present invention, there is provided a process for identifying a target double-stranded nucleic acid in a sample comprising the steps of:
(i) providing a sample in which a target single-stranded nucleic acid may exist;
(ii) mixing the sample with the above pyrylium compound;
(iii) irradiating the sample resulting from the step (ii) with light having a wavelength which is substantially equal to the maximum absorption wavelength when the pyrylium compound is intercalated into the double-stranded nucleic acid to excite the intercalated pyrylium compound; and
(iv) ascertaining emission of fluorescence from the excited pyrylium compound to identify whether or not the target double-stranded nucleic acid exists in the sample.
Further objects, features and advantages of the present invention will become apparent from the following description of the preferred embodiments with reference to the attached drawings.